Synthetic Biology Toolkit for a New Species of Pseudomonas Promissory for Electricity Generation in Microbial Fuel Cells.

Microbial fuel cells (MFCs) offer sustainable solutions for various biotechnological applications and are a crucial area of research in biotechnology. MFCs can effectively treat various refuse, such as wastewater and biodiesel waste by decomposing organic matter and generating electricity. Certain Pseudomonas species possess extracellular electron transfer (EET) pathways, enabling them to transfer electrons from organic compounds to the MFC's anode. Moreover, Pseudomonas species can grow under low-oxygen conditions, which is advantageous considering that the electron transfer process in an MFC typically leads to reduced oxygen levels at the anode. This study focuses on evaluating MFCs inoculated with a new Pseudomonas species grown with 1 g.L-1 glycerol, a common byproduct of biodiesel production. Pseudomonas sp. BJa5 exhibited a maximum power density of 39 mW.m-2. Also, the observed voltammograms and genome analysis indicate the potential production of novel redox mediators by BJa5. Additionally, we investigated the bacterium's potential as a synthetic biology non-model chassis. Through testing various genetic parts, including constitutive promoters, replication origins and cargos using pSEVA vectors as a scaffold, we assessed the bacterium's suitability. Overall, our findings offer valuable insights into utilizing Pseudomonas spp. BJa5 as a novel chassis for MFCs. Synthetic biology approaches can further enhance the performance of this bacterium in MFCs, providing avenues for improvement.

Enhancing 1,3-Propanediol Productivity in the Non-Model Chassis Clostridium beijerinckii through Genetic Manipulation.

Biotechnological processes at biorefineries are considered one of the most attractive alternatives for valorizing biomasses by converting them into bioproducts, biofuels, and bioenergy. For example, biodiesel can be obtained from oils and grease but generates glycerol as a byproduct. Glycerol recycling has been studied in several bioprocesses, with one of them being its conversion to 1,3-propanediol (1,3-PDO) by Clostridium. Clostridium beijerinckii is particularly interesting because it can produce a range of industrially relevant chemicals, including solvents and organic acids, and it is non-pathogenic. However, while Clostridium species have many potential advantages as chassis for synthetic biology applications, there are significant limitations when considering their use, such as their limited genetic tools, slow growth rate, and oxygen sensitivity. In this work, we carried out the overexpression of the genes involved in the synthesis of 1,3-PDO in C. beijerinckii Br21, which allowed us to increase the 1,3-PDO productivity in this strain. Thus, this study contributed to a better understanding of the metabolic pathways of glycerol conversion to 1,3-PDO by a C. beijerinckii isolate. Also, it made it possible to establish a transformation method of a modular vector in this strain, therefore expanding the limited genetic tools available for this bacterium, which is highly relevant in biotechnological applications.

Electrobiochemical skills of Pseudomonas aeruginosa species that produce pyocyanin or pyoverdine for glycerol oxidation in a microbial fuel cell.

Pseudomonas aeruginosa can produce pigments, which mediate external electron transfer (EET). Depending on the mediator, this species can be explored in bioelectrosystems to harvest energy or to obtain chemicals from residual organic compounds. This study has compared the performance of microbial fuel cells (MFCs) inoculated with a Pseudomonas aeruginosa isolate, namely EW603 or EW819, which produce pyocyanin and pyoverdine, respectively. The efficiency of these MFCs in glycerol, a typical residue of biodiesel production, were also compared. The MFCs exhibited different performances. The maximum voltage was 411 and 281 mV m2, the power density was 40.1 and 21.3 mW m-2, and the coulombic efficiency was 5.16 and 1.49% for MFC-EW603 and MFC-EW819, respectively. MFC-EW603 and MFC-EW819 achieved maximum current at 560 and 2200 Ω, at 141.2 and 91.3 mA m-2, respectively. When the system was operated at the respective maximum current output, MFC-EW603 consumed the total glycerol content (11 mmol L-1), and no products could be detected after 50 h. In turn, acetic and butyric acids were detected at the end of MFC-EW819 operation (75 h). The results suggested that P. aeruginosa metabolism can be steered in the MFC to generate current or microbial products depending on the pigment-producing strain and the conditions applied to the system, such as the external resistance. In addition, gene cluster pathways related to phenazine production (phzA and phzB) and other electrogenic-related genes (mexGHI-opmB) were identified in the strain genomes, supporting the findings. These results open new possibilities for using glycerol in bioelectrochemical systems.

Ideal conditions of microwave-assisted acid pretreatment of sugarcane straw allow fermentative butyric acid production without detoxification step.

Sugarcane straw (SCS) was pretreated with dilute sulfuric acid assisted by microwave to magnify fermentable sugars and to minimize the concentration of inhibitors in the hydrolysates. The optimum conditions for maximum recovery of sugars were 162 °C and 0.6% (w/v) H2SO4. The low level of inhibitors, such as acetate (2.9 g/L) and total phenolics (1.4 g/L), in the SCS slurry from the pretreatment stage allowed the enzymatic hydrolysis and fermentation steps to occur without detoxification. Besides consuming the total sugar content (31.0 g/L), Clostridium beijerinckii Br21 was able to use acetate from the SCS hydrolysate, to give butyric acid at high conversion factor (0.49 g of butyric acid /g of sugar). The optimized pretreatment conditions spared acid, time, and the detoxification stage, making bio-butyric acid production from SCS extremely attractive.

Genome sequence of the H2-producing Clostridium beijerinckii strain Br21 isolated from a sugarcane vinasse treatment plant.

We report on the nearly complete genome sequence of Clostridium beijerinckii strain Br21, formerly isolated from a sugarcarne vinasse wastewater treatment plant. The resulting genome is ca. 5.9 Mbp in length and resembles the size of previously published C. beijerinckii genomes. We annotated the genome sequence and predicted a total of 5323 genes. Strain Br21 has a genetic toolkit that allows it to exploit diverse sugars that are often found after lignocellulosic biomass pretreatment to yield products of commercial interest. Besides the whole set of genes encoding for enzymes underlying hydrogen production, the genome of the new strain includes genes that enable carbon sources conversion into butanol, ethanol, acetic acid, butyric acid, and the chemical block 1,3-propanediol, which is used to obtain polymers. Moreover, the genome of strain Br21 has a higher number of ORFs with predicted beta-glucosidase activity as compared to other C. beijerinckii strains described in the KEGG database. These characteristics make C. beijerinckii strain Br21 a remarkable candidate for direct use in biotechnological processes and attest that it is a potential biocatalyst supplier.

Hydrogen and electrical energy co-generation by a cooperative fermentation system comprising Clostridium and microbial fuel cell inoculated with port drainage sediment.

This research work has succeeded in recovering energy from glucose by generating H2 with the aid of a Clostridium beijerinckii strain and obtaining electrical energy from compounds present in the H2 fermentation effluent in a microbial fuel cell (MFC) seeded with native port drainage sediment. In the fermentation step, 49.5% of the initial glucose concentration (56 mmol/L) was used to produce 104 mmol/L H2; 5, 33, 3, and 1 mmol/L acetate, butyrate, lactate, and ethanol also emerged, respectively. MFC tests by feeding the anodic compartment with acetate, butyrate, lactate (individually or as a mixture), or the H2 fermentation effluent provided power density values ranging between 0.6 and 1.2 W/m2. Acetate furnished the highest power density with a nanowire-rich biofilm despite the lowest anode bacterial concentration (1012 16S gene copies/g of sediment). Non-conventional exoelectrogenic microbial communities were observed in the acetate-fed MFC; e.g., Pseudomonadaceae (Pseudomonas) and Clostridia (Acidaminobacter, Fusibacter).

Fermentative hydrogen production from agroindustrial lignocellulosic substrates.

To achieve economically competitive biological hydrogen production, it is crucial to consider inexpensive materials such as lignocellulosic substrate residues derived from agroindustrial activities. It is possible to use (1) lignocellulosic materials without any type of pretreatment, (2) lignocellulosic materials after a pretreatment step, and (3) lignocellulosic materials hydrolysates originating from a pretreatment step followed by enzymatic hydrolysis. According to the current literature data on fermentative H2 production presented in this review, thermophilic conditions produce H2 in yields approximately 75% higher than those obtained in mesophilic conditions using untreated lignocellulosic substrates. The average H2 production from pretreated material is 3.17 ± 1.79 mmol of H2/g of substrate, which is approximately 50% higher compared with the average yield achieved using untreated materials (2.17 ± 1.84 mmol of H2/g of substrate). Biological pretreatment affords the highest average yield 4.54 ± 1.78 mmol of H2/g of substrate compared with the acid and basic pretreatment - average yields of 2.94 ± 1.85 and 2.41 ± 1.52 mmol of H2/g of substrate, respectively. The average H2 yield from hydrolysates, obtained from a pretreatment step and enzymatic hydrolysis (3.78 ± 1.92 mmol of H2/g), was lower compared with the yield of substrates pretreated by biological methods only, demonstrating that it is important to avoid the formation of inhibitors generated by chemical pretreatments. Based on this review, exploring other microorganisms and optimizing the pretreatment and hydrolysis conditions can make the use of lignocellulosic substrates a sustainable way to produce H2.

Fermentative hydrogen production from agroindustrial lignocellulosic substrates

To achieve economically competitive biological hydrogen production, it is crucial to consider inexpensive materials such as lignocellulosic substrate residues derived from agroindustrial activities. It is possible to use (1) lignocellulosic materials without any type of pretreatment, (2) lignocellulosic materials after a pretreatment step, and (3) lignocellulosic materials hydrolysates originating from a pretreatment step followed by enzymatic hydrolysis. According to the current literature data on fermentative H2 production presented in this review, thermophilic conditions produce H2 in yields approximately 75% higher than those obtained in mesophilic conditions using untreated lignocellulosic substrates. The average H2 production from pretreated material is 3.17 ± 1.79 mmol of H2/g of substrate, which is approximately 50% higher compared with the average yield achieved using untreated materials (2.17 ± 1.84 mmol of H2/g of substrate). Biological pretreatment affords the highest average yield 4.54 ± 1.78 mmol of H2/g of substrate compared with the acid and basic pretreatment - average yields of 2.94 ± 1.85 and 2.41 ± 1.52 mmol of H2/g of substrate, respectively. The average H2 yield from hydrolysates, obtained from a pretreatment step and enzymatic hydrolysis (3.78 ± 1.92 mmol of H2/g), was lower compared with the yield of substrates pretreated by biological methods only, demonstrating that it is important to avoid the formation of inhibitors generated by chemical pretreatments. Based on this review, exploring other microorganisms and optimizing the pretreatment and hydrolysis conditions can make the use of lignocellulosic substrates a sustainable way to produce H2.

Rapid analysis for predicting the expansion of the polvilho azedo

Sour cassava starch (polvilho azedo) is a chemically and enzymatically modified starch. It is used to prepare the traditional baked goods. Because of its satisfactory expansion property, a light and alveolar structure is produced in baked food prepared with sour cassava starch, without adding any chemical or biological ferment. This study reports some easy and simple procedures for pre-estimating sour cassava starch expansion property.The correlation between the native cassava starches and the laboratory-produced sour cassava starch was analyzed. An investigation among the local bakers was carried out for inquiring into the polvilho azedo choice,and it disclosed some product brands. The polvilho azedo samples from these brands were also evaluated. Acid factor, pH, specific volume, swelling power and intrinsic viscosity characteristics were directly correlated with expansion property. The procedures for determining acid factor, pH and weight loss are easy and accessible methodologies which might be carried out at small industries and bakeries.

Caracterização da microfauna em estação de tratamento de esgotos do tipo lodos ativados: um instrumento de avaliação e controle do processo / Microfauna characterization in activated sludge wastewater treatment plant: an instrument of assessment and control of process

A microfauna presente em uma ETE por lodos ativados foi caracterizada, correlacionada com parâmetros físico-químicos e utilizada em modelos existentes de avaliação de sistemas. Durante 351 dias o lodo ativado manteve-se estável com flocos compactos, pequenos e com poucos filamentos. Os protozoários predominantes no lodo foram as tecamebas e os ciliados predadores de flocos, indicando estabilidade do sistema. Os métodos propostos em literatura, baseados no IBL, na densidade total da microfauna e na relação entre ciliados predadores de flocos e ciliados fixos, mostraram-se eficientes para o diagnóstico das condições depurativas do sistema. A análise qualitativa do lodo, englobando o tamanho, quantidade de filamentos, compactação e a identificação das espécies dominantes, foi suficiente para uma caracterização imediata das condições depurativas do sistema de tratamento.